RESUMO
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, mainly associated with liver cirrhosis. Current diagnostic methods for HCC have limited sensitivity and specificity, highlighting the need for improved early detection and intervention. In this study, we used a comprehensive approach involving endogenous peptidome along with bioinformatics analysis to identify and evaluate potential biomarkers for HCC. Serum samples from 40 subjects, comprising 20 HCC cases and 20 patients with liver cirrhosis (CIRR), were analyzed. Among 2568 endogenous peptides, 67 showed significant differential expression between the HCC vs CIRR. Further analysis revealed three endogenous peptides (VMHEALHNHYTQKSLSLSPG, NRFTQKSLSLSPG, and SARQSTLDKEL) that showed far better performance compared to AFP in terms of area under the receiver operating characteristic curve (AUC), showcasing their potential as biomarkers for HCC. Additionally, endogenous peptide IAVEWESNGQPENNYKT that belongs to the precursor protein Immunoglobulin heavy constant gamma 4 was detected in 100% of the HCC group and completely absent in the CIRR group, suggesting a promising diagnostic biomarker. Gene ontology and pathway analysis revealed the potential involvement of these dysregulated peptides in HCC. These findings provide valuable insights into the molecular basis of HCC and may contribute to the development of improved diagnostic methods and therapeutic targets for HCC.
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In this work, a composite hydrogel material consisting of chitosan-based composite hydrogel was prepared by a simple and rapid synthetic method and will be named three-dimensional (3D)-IL-COF-1@CS hydrogel. Possessing a stable 3D network structure and outstanding hydrophilicity, the novel hydrogel is capable of capturing glycopeptides. The 3D-IL-COF-1@CS hydrogel showed good sensitivity (0.1 fmol/µL) and selectivity (1:2000). In addition, 19 glycopeptides were captured in standard samples. In the analysis of human serum, 148 glycopeptides assigned to 72 glycoproteins were assayed in the serum of normal individuals, and 245 glycopeptides corresponding to 100 glycoproteins were found in the serum of colorectal cancer (CRC) patients. More importantly, several functional programs based on Gene Ontology analysis supported molecular biological processes that may be relevant to the pathogenesis of CRC, including aging, fibrinogen complex, and arylesterase activity. The low cost, simplicity, rapid synthesis, and good enrichment performance have a great future in glycoproteomics analysis and related diseases.
Assuntos
Neoplasias Colorretais , Glicopeptídeos , Interações Hidrofóbicas e Hidrofílicas , Humanos , Neoplasias Colorretais/sangue , Glicopeptídeos/sangue , Glicopeptídeos/química , Hidrogéis/química , Polímeros/química , Quitosana/químicaRESUMO
A porphyrin-based titanium-rich porous organic polymer (Th-PPOPs@Ti4+) was designed based on immobilized metal ion affinity chromatography technique and successfully applied to phosphopeptide enrichment with 5,10,15,20-tetrakis(4-carboxyphenyl) porphine tetramethyl ester (TCPTE), 2,3-dihydroxyterephthalaldehyde (DHTA), and 2,3,4-trihydroxybenzaldehyde (THBA) as raw materials. Th-PPOPs@Ti4+ exhibited remarkable sensitivity (0.5 fmol), high selectivity (ß-casein: BSA = 1:2000, molar ratio), outstanding recovery (95.0 ± 1.9%), reusability (10 times), and superior loading capacity (143 mg·g-1). In addition, Th-PPOPs@Ti4+ exhibited excellent ability to specifically capture phosphopeptides from the serum of colorectal cancer (CRC) individuals and normal subjects. Sixty phosphopeptides assigned to 35 phosphoproteins were obtained from the serum of CRC individuals, and 43 phosphopeptides allocated to 28 phosphoproteins were extracted in the serum of healthy individuals via nano-LC-MS/MS. Gene ontology assays revealed that the detected phosphoproteins may be inextricably tied to CRC-associated events, including response to estrogen, inflammatory response, and heparin binding, suggesting that it is possible that these correlative pathways may be implicated in the pathogenesis of CRC.
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Neoplasias Colorretais , Fosfopeptídeos , Porfirinas , Titânio , Humanos , Neoplasias Colorretais/sangue , Titânio/química , Fosfopeptídeos/sangue , Fosfopeptídeos/isolamento & purificação , Fosfopeptídeos/química , Porosidade , Porfirinas/química , Polímeros/químicaRESUMO
Protein glycosylation of human serum exosomes can reveal significant physiological information, and the development of large-scale identification strategies is crucial for the in-depth investigation of the serum exosome glycoproteome. In this study, using surface functionalization techniques, an ultra-hydrophilic mesoporous silica magnetic nanosphere (denoted as Fe3O4-CG@mSiO2) was synthesized for the quick and accurate detection of glycopeptides from HRP digests. The Fe3O4-CG@mSiO2 nanospheres demonstrated outstanding enrichment capability, high sensitivity (5 amol/µL), good size exclusion effect (HRP digests/BSA proteins, 1:10,000), stable reusability (at least 10 times), and an excellent recovery rate (108.6 ± 5.5%). Additionally, after enrichment by Fe3O4-CG@mSiO2, 156 glycopeptides assigned to 64 proteins derived from human serum exosomes were successfully identified, which demonstrates that the nanospheres have great potential for the research of the large-scale serum exosome glycoproteome.
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Exossomos , Glicopeptídeos , Humanos , Dióxido de Silício , Magnetismo , Interações Hidrofóbicas e Hidrofílicas , Proteoma , Fenômenos MagnéticosRESUMO
Pluripotent stem cells (PSC) endocrine differentiation at a large scale allows sampling of transcriptome and proteome with phosphoproteome (proteoform) at specific time points. We describe the dynamic time course of changes in cells undergoing directed beta-cell differentiation and show target proteins or previously unknown phosphorylation of critical proteins in pancreas development, NKX6-1, and Chromogranin A (CHGA). We describe fluctuations in the correlation between gene expression, protein abundance, and phosphorylation, following differentiation protocol perturbations at all stages to identify proteoform profiles. Our modeling recognizes outliers on a phenomic landscape of endocrine differentiation, and we describe new biological pathways involved. We have validated our proteomic data by analyzing independent single-cell RNAseq datasets for in-vitro pancreatic islet production and corroborated our findings for several proteins suggestive as targets for future research. The single-cell analysis combined with proteoform data places new protein targets within the specific time point and at the specific pancreatic lineage of differentiating stem cells. We suggest that non-correlating proteins abundances or new phosphorylation motifs of NKX6.1 and CHGA point to new signaling pathways that may play an essential role in beta-cell development. We present our findings for the research community's use to improve endocrine differentiation protocols and developmental studies.
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Células-Tronco Embrionárias Humanas , Células-Tronco Pluripotentes , Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes/metabolismo , ProteômicaRESUMO
Ribosome dimerization is one of the bacterial events that suppresses protein synthesis in the stationary phase. Protein factors responsible for ribosome dimerization in bacteria are well characterized, whereas no information is available for the corresponding factors in archaeal and eukaryotic cells. Here we describe a protein found among the ribosome-associated proteins which dimerizes the 30S ribosomal subunit of the archaeon Pyrococcus furiosus. The ribosome-associated proteins were prepared by high-salt wash of crude ribosomes, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Of the detected proteins we focused on a protein (PF0560) whose Protein Score was the highest of all of the function-unknown proteins. PF0560 protein had a pronounced effect on the sedimentation pattern of the 30S ribosomal subunit; addition of this protein to isolated 30S subunit reduced the 30S fraction and increased the amount of the 50S fraction. This increase presumably corresponds to the dimer of the 30S subunit. The PF0560-dependent 30S-dimerization, was also observed by gel electrophoretic analysis. This effect was not observed in EDTA-treated 30S subunit, with protein-free 16S rRNA or with bacterial/eukaryotic ribosomal small subunits. Furthermore, PF0560 protein suppressed the formation of functional 70S ribosomes. These results suggest that PF0560 is a novel 30S dimerization factor, which might participate in regulation of archaeal translation.
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Proteínas Arqueais/metabolismo , Dimerização , Proteoma/metabolismo , Pyrococcus furiosus/metabolismo , RNA Ribossômico 16S/química , Proteínas Ribossômicas/metabolismo , Ribossomos/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Magnésio/química , Proteoma/análise , Pyrococcus furiosus/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/genética , Homologia de SequênciaRESUMO
A sequential design strategy was applied to optimize the secretion of pectinases by a Saccharomyces cerevisiae strain, from Brazilian sugarcane liquor vat, on passion fruit residue flour (PFRF), through solid-state fermentation (SSF). A factorial design was performed to determine the influence variables and two rotational central composite designs were executed. The validated experimental result was of 7.1 U mL-1 using 50% PFRF (w/w), pH 5, 30 °C for 24 h, under static SSF. Polygalacturonase, pectin methyl esterase, pectin-lyase and pectate-lyase activities were 3.5; 0.08; 3.1 and 0.8 U mL-1, respectively. Shotgun proteomics analysis of the crude extract enabled the identification of two pectin-lyases, one pectate-lyase and a glucosidase. The crude enzymatic extract maintained at least 80% of its original activity at pH values and temperatures ranging from 2 to 8 and 30 to 80 °C, respectively, over 60 min incubation. Results revealed that PFRF might be a cost-effective and eco-friendly substrate to produce pectinases. Statistical optimization led to fermentation conditions wherein pectin active proteins predominated. To the extent of our knowledge, this is the first study reporting the synthesis of pectate lyase by S. cerevisiae.
Assuntos
Poligalacturonase , Saccharomyces cerevisiae , Fermentação , Concentração de Íons de Hidrogênio , Pectinas/metabolismo , Poligalacturonase/metabolismo , Proteômica , Saccharomyces cerevisiae/metabolismoRESUMO
AIM: Endometriosis is a debilitating disease marked by recurrent gynecological proliferations. The present study aimed at performing differential proteomic analysis of matched eutopic and ectopic endometrium from women with ovarian endometriosis. MATERIALS AND METHODS: Proteomes were resolved using nano LC-MS and further identified and quantified using ProteinLynx Global SERVER (PLGS) software. Selected proteins were further chosen for validation by real time-polymerase chain reaction (RT-PCR). RESULTS: The protein profiles uncovered several differentially expressed proteins in the diseased sample (ectopic endometrium) as compared to the reference sample (eutopic endometrium). The study involved an advanced proteomic approach, nano LC-MS, and validates for the first time the upregulation of Mimecan and Lumican proteins in endometriosis. CONCLUSIONS: These proteins may hence prove as potentially useful tools in the search for diagnostic markers for early detection of the disease.
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Anexina A5/metabolismo , Endometriose/metabolismo , Endométrio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lumicana/metabolismo , Doenças Ovarianas/metabolismo , Transferrina/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , ProteomaRESUMO
Cannabis sativa L. is an herbaceous plant belonging to the family of Cannabaceae. It is classified into three different chemotypes based on the different cannabinoids profile. In particular, fiber-type cannabis (hemp) is rich in cannabidiol (CBD) content. In the present work, a rapid nano liquid chromatographic method (nano-LC) was proposed for the determination of the main cannabinoids in Cannabis sativa L. (hemp) inflorescences belonging to different varieties. The nano-LC experiments were carried out in a 100 µm internal diameter capillary column packed with a C18 stationary phase for 15 cm with a mobile phase composed of ACN/H2O/formic acid, 80/19/1% (v/v/v). The reverse-phase nano-LC method allowed the complete separation of four standard cannabinoids in less than 12 min under isocratic elution mode. The nano-LC method coupled to ultraviolet (UV) detection was validated and applied to the quantification of the target analytes in cannabis extracts. The nano-LC system was also coupled to an electrospray ionization-mass spectrometry (ESI-MS) detector to confirm the identity of the cannabinoids present in hemp samples. For the extraction of the cannabinoids, three different approaches, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), and an extraction procedure adapted from the French Pharmacopeia's protocol on medicinal plants, were carried out, and the results achieved were compared.
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Canabinoides/análise , Cannabis/química , Cromatografia Líquida , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Advancements in proteomic tools offer a comprehensive solution to studying the complexity of diseases at molecular level. This study focusses on the clinical proteomic profiling of pre- and post-hydroxyurea (HU)-treated ß-thalassemia patients in parallel with healthy individuals to better understand the role of HU in the treatment of ß-thalassemia. The strategy encompasses sequential high-resolution protein fractionation using MicroSol-isoelectric focusing (ZOOM- IEF) followed by one-dimensional SDS-PAGE before nano-RP-LC-MS/ MS analysis of tryptic peptides. Protein identification was performed through Mascot search using NCBInr and SwissProt databases. Several different proteins were observed in pool serum samples of each of the three study groups. Approximately, 1250 proteins exclusive to each group were identified, and after removing the redundant and low sequence coverage proteins, the number was reduced to 576 (201 in healthy, 187 in HU-untreated and 188 in HU-treated group). Uniquely identified proteins in the HU-treated group regulate the focal adhesion, ECM-receptor interaction, PI3K-Akt signaling, Rap1 signaling, cAMP signaling, platelet activation, and Ca2+ signaling pathways in the HU-treated group. The proteomic profile presented here will add to the current state of understanding of molecular mechanisms involved in hydroxyurea treatment of ß-thalassemia.
Assuntos
Proteínas Sanguíneas/análise , Focalização Isoelétrica/métodos , Proteoma/análise , Proteômica/métodos , Talassemia beta , Biomarcadores/análise , Proteínas Sanguíneas/química , Proteínas Sanguíneas/classificação , Proteínas Sanguíneas/isolamento & purificação , Cromatografia Líquida/métodos , Humanos , Hidroxiureia/química , Nanomedicina , Proteoma/química , Proteoma/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Talassemia beta/sangue , Talassemia beta/metabolismoRESUMO
Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in fetal labyrinth trophoblasts as part of the feto-maternal barrier, necessary for sufficient calcium supply, embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact labyrinth morphology of Trpv6-deficient placentae, and reduced Ca2+ uptake of primary trophoblasts upon functional deletion of TRPV6. Trpv6-/- trophoblasts show a distinct calcium-dependent phenotype. Deep proteomic profiling of wt and Trpv6-/- primary trophoblasts using label-free quantitative mass spectrometry leads to the identification of 2778 proteins. Among those, a group of proteases, including high-temperature requirement A serine peptidase 1 (HTRA1) and different granzymes are more abundantly expressed in Trpv6-/- trophoblast lysates, whereas the extracellular matrix protein fibronectin and the fibronectin-domain-containing protein 3A (FND3A) were markedly reduced. Trpv6-/-placenta lysates contain a higher intrinsic proteolytic activity increasing fibronectin degradation. Our results show that the extracellular matrix formation of the placental labyrinth depends on TRPV6; its deletion in trophoblasts correlates with the increased expression of proteases controlling the extracellular matrix in the labyrinth during pregnancy.
Assuntos
Matriz Extracelular/metabolismo , Placenta/metabolismo , Canais de Cátion TRPV/metabolismo , Transporte Biológico , Biomarcadores , Cálcio/metabolismo , Movimento Celular/genética , Sobrevivência Celular/genética , Biologia Computacional , Feminino , Técnicas de Silenciamento de Genes , Humanos , Gravidez , Proteólise , Proteoma , Proteômica , Canais de Cátion TRPV/genéticaRESUMO
Improvements in mass spectrometry technology to include instrument duty cycle, resolution, and sensitivity suggest mass spectrometry as a highly competitive alternative to conventional microbiological proteomic techniques. Targeted mass spectral analysis, sans prior empirical measurements, has begun to solely use the enormous amount of available genomic information for assay development. An in silico tryptic digestion of a suspected antibiotic-resistant enzyme using only its genomic information for assay development was achieved. Both MRM and full-scan MS2 independent data acquisitions were obtained for an antibiotic-resistance microbe not previously measured using mass spectrometry. In addition, computation methods to determine highest responding peptides in positive ion mode liquid chromatography-mass spectrometry (LC-MS) were evaluated. Employment of the relative retention time (iRT) concept using a homemade peptide standard set revealed facile method transfer between two fundamental different mass spectral platforms: an ultra-high-pressure liquid chromatography triple quadrupole-mass spectrometer (UHPLC-MS) and nano-liquid chromatography parallel reaction monitoring (nano-LC-PRM) hybrid quadrupole orbitrap Q-exactive mass spectrometer supporting easy dissemination and rapid method implementation between laboratories. Graphical Abstract.
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Canamicina Quinase/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Cromatografia Líquida/métodos , Canamicina Quinase/química , Limite de Detecção , Espectrometria de Massas/métodosRESUMO
Protein-protein interactions (PPIs) lead the formation of protein complexes that perform biochemical reactions that maintain the living state of the living cell. Although therapeutic drugs should influence the formation of protein complexes in addition to PPI network, the methodology analyzing such influences remain to be developed. Here, we demonstrate that a new approach combining HPLC (high performance liquid chromatography) for separating protein complexes, and the SILAC (stable isotope labeling using amino acids in cell culture) method for relative protein quantification, enable us to identify the protein complexes influenced by a drug. We applied this approach to the analysis of thalidomide action on HepG2 cells, assessed the identified proteins by clustering data analyses, and assigned 135 novel protein complexes affected by the drug. We propose that this approach is applicable to elucidating the mechanisms of actions of other therapeutic drugs on the PPI network, and the formation of protein complexes.
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Aminoácidos/química , Avaliação Pré-Clínica de Medicamentos/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas/química , Proteínas/metabolismo , Proteômica , Células Hep G2 , Humanos , Marcação por Isótopo , Talidomida/farmacologiaRESUMO
In this work, for the first time, a liquid open tubular column modified by five-layer gold nanoparticles and linked with C18 (GNPs@C18 ) was designed and fabricated for nano-LC-MS/MS analysis of 80 living cells. Sixty nanometer gold nanoparticles were self-assembled layer by layer on the inner wall of a 20 µm id fused-silica capillary. C18 was then linked on the gold nanoparticles to make the liquid open tubular column show hydrophobic character. Enough loading capacities for analysis of 80 living cells, â¼100 fmol for pk-10 and â¼30 fmol for insulin, were obtained with the 2 m × 20 µm id five-layer GNPs@C18 open tubular column. The open tubular column was used in an online pretreatment and direct nano-LC-MS/MS analysis system to analyze 80 living HepG2 cells. In total, 650 proteins were identified in triplicate runs. The subcellular localization of the identified proteins showed that our system had no bias toward different cellular compartments. Protein copy number per cell of the identified proteins showed that the detection limit could reach 50 zmol and the abundance of the identified proteins could cover a dynamic range of 6 orders.
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Cromatografia Líquida/instrumentação , Nanopartículas/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Ouro/química , Células Hep G2 , Humanos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Proteômica/instrumentação , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
Prostate cancer is the leading type of cancer diagnosed, and the most frequent cause of worldwide male cancer-related deaths annually. The limitations of current prostate cancer screening tests demand the identification of novel biomarkers for the early diagnosis of prostate cancer bone metastasis. In the present study, we performed a proteomic analysis of secreted proteins from the prostate cancer bone metastasis cell line, PC-3, and the normal prostate cell line, RWPE-1. We thus quantified 917 proteins, of which 68 were found to be secreted at higher levels by PC-3 than by RWPE-1 cells via LC-MS/MS. To characterize the highly secreted proteins in the PC-3 cell line and thereby identify biomarker proteins, we divided the quantifiable proteins into four quantitative categories (Q1-Q4). The KEGG lysine degradation and osteoclast differentiation pathways were demonstrated to be enriched in the highly secreted Q4 protein group. Transforming growth factor (TGF) beta family proteins related to osteoclast differentiation were identified as key regulators of PC-3 cell proliferation. Immunoblotting was used to confirm the observed high level of pentraxin, follistatin, TGF-beta family members, and serpin B3 secretion by PC-3 cells. From the collective results of the present study, we suggest that serpin B3 is a promising novel biomarker candidate for the diagnosis of prostate cancer bone metastasis.
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Biomarcadores Tumorais/análise , Neoplasias Ósseas/diagnóstico , Neoplasias da Próstata/diagnóstico , Proteoma/análise , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Proliferação de Células , Cromatografia Líquida , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteoma/metabolismo , Proteômica , Serpinas/análise , Serpinas/metabolismo , Espectrometria de Massas em TandemRESUMO
Dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides were rapidly identified from Ruditapes philippinarum hydrolysate. The hydrolysate was fractionated by ethanol precipitation and preparative reverse phase high-performance liquid chromatography (RP-HPLC). The fraction which showed the highest DPP-IV inhibitory activity was then analyzed by a high-throughput nano-liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC ESI-MS/MS) method, and the sequences of peptides were identified based on the MS/MS spectra against the Mollusca protein data from the UniProt database. In total, 50 peptides were identified. Furthermore, molecular docking was used to identify potential DPP-IV inhibitors from the identified peptides. Docking results suggested that four peptides: FAGDDAPR, LAPSTM, FAGDDAPRA, and FLMESH, could bind pockets of DPP-IV through hydrogen bonds, π-π bonds, and charge interactions. The four peptides were chemically synthesized and tested for DPP-IV inhibitory activity. The results showed that they possessed DPP-IV inhibitory activity with IC50 values of 168.72 µM, 140.82 µM, 393.30 µM, and >500 µM, respectively. These results indicate that R. philippinarum-derived peptides may have potential as functional food ingredients for the prevention of diabetes.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Peptídeos/farmacologia , Sequência de Aminoácidos/genética , Animais , Bivalves/química , Cromatografia de Fase Reversa , Diabetes Mellitus/patologia , Inibidores da Dipeptidil Peptidase IV/química , Humanos , Simulação de Acoplamento Molecular , Peptídeos/química , Espectrometria de Massas em TandemRESUMO
Panax ginseng is an important herb that has clear effects on the treatment of diverse diseases. Until now, the natural peptide constitution of this herb remains unclear. Here, we conduct an extensive characterization of Ginseng peptidome using MS-based data mining and sequencing. The screen on the charge states of precursor ions indicated that Ginseng is a peptide-rich herb in comparison of a number of commonly used herbs. The Ginseng peptides were then extracted and submitted to nano-LC-MS/MS analysis using different fragmentation modes, including CID, high-energy collisional dissociation, and electron transfer dissociation. Further database search and de novo sequencing allowed the identification of total 308 peptides, some of which might have important biological activities. This study illustrates the abundance and sequences of endogenous Ginseng peptides, thus providing the information of more candidates for the screening of active compounds for future biological research and drug discovery studies.
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Panax/genética , Peptídeos/genética , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Panax/química , Peptídeos/química , Peptídeos/classificação , Peptídeos/isolamento & purificaçãoRESUMO
A newly synthesized acridone derivative 8a shows potent antitumor activity against CCRF-CEM leukemia cells. Herein, the first proteomic study of 8a effects in CCRF-CEM cells was performed by 2D nano-LC-ESI-MS/MS to better understand the mechanisms of action of 8a. Data analyses based on PLGS, STRING, Cytoscape, and database for annotation, visualization, and integrated discovery identified 55 proteins that were differentially expressed in response to 8a exposure. Multiple cellular pathways were affected, including chromatin organization, energy metabolism, DNA repair, oxidative-stress, and apoptosis. The changes in protein expression were further verified for PKM2. Moreover, 8a lowered down the expression of HEX and PFK-1. Lactate production was decreased in 8a-treated cells, indicating suppression of glycolysis. The elevated XRCC6 and decreased histone expression levels suggested increased DNA damage in 8a-treated cells, which was confirmed by the increased γ-H2AX foci. Molecular docking of 8a with DNA demonstrated direct interactions of 8a with DNA through three hydrogen bonds and four π-π interactions, potentially explaining the mode of action that 8a damaged to DNA. The differential protein profiling and dysfunction of metabolic pathways induced by 8a provide novel insights into the potential action mechanisms of 8a.
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Acridonas/toxicidade , Apoptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Metabolismo Energético/efeitos dos fármacos , Humanos , Espectrometria de MassasRESUMO
The study gives an overview of the tests and analyses undertaken in the past 20 years to establish the presence of blood in the foundation layers of Chinese lacquer artefacts and also shows the development of analytical methods over that period. When undertaking the conservation of lacquer objects it is crucial to know the type of binding medium as this influences the selection of any consolidants that may be required in the treatment. Microchemical tests to identify blood using benzidine and luminol, various chromatographic and mass spectrometric techniques and DNA analyses were assessed on selected Chinese lacquer objects, and the results gained are summarized.
RESUMO
Human cardiac stem cells (hCSC) express a portfolio of plasma membrane receptors that are involved in the regulatory auto/paracrine feedback loop mechanism of activation of these cells, and consequently contribute to myocardial regeneration. In order to attain a comprehensive description of hCSC receptome and overcoming the inability demonstrated by other technologies applied in receptor identification, mainly due to the transmembrane nature, high hydrophobic character and relative low concentration of these proteins, we have exploited and improved a proteomics workflow. This approach was based on the enrichment of hCSC plasma membrane fraction and addition of prefractionation steps prior to MS analysis. More than 100 plasma membrane receptors were identified. The data reported herein constitute a valuable source of information to further understand cardiac stem cells activation mechanisms and the subsequent cardiac repair process. All MS data have been deposited in the ProteomeXchange with identifier PXD001117 (http://proteomecentral.proteomexchange.org/dataset/PXD001117).